Mutation of Escherichia coli cytosine deaminase significantly enhances molecular chemotherapy of human glioma

Abstract

Combined treatment using adenoviral (Ad)-directed enzyme/ prodrug therapy and radiation therapy has the potential to become a powerful method of cancer therapy. We have developed an Ad vector encoding a mutant bacterial cytosine deaminase (bCD) gene (AdbCD-D314A), which has a higher affinity for cytosine than wild-type bCD (bCDwt). The purpose of this study was to evaluate cytotoxicity in vitro and therapeutic efficacy in vivo of the combination of AdbCD-D314A with the prodrug 5-fluorocytosine (5-FC) and ionizing radiation against human glioma. The present study demonstrates that AdbCD-D314A infection resulted in increased 5-FC-mediated cell killing, compared with AdbCDwt. Furthermore, a significant increase in cytotoxicity following AdbCD-D314A and radiation treatment of glioma cells in vitro was demonstrated as compared to AdbCDwt. Animal studies showed significant inhibition of subcutaneous or intracranial tumor growth of D54MG glioma xenografts by the combination of AdbCD-D314A/5-FC with ionizing radiation as compared with either agent alone, and with AdbCDwt/5-FC plus radiation. The results suggest that the combination of AdbCD-D314A/5-FC with radiation produces markedly increased cytotoxic effects in cancer cells in vitro and in vivo. These data indicate that combined treatment with this novel mutant enzyme/prodrug therapy and radiotherapy provides a promising approach for cancer therapy.

Enzyme/Prodrug Systems for Cancer Gene Therapy

“…Since 5-FU traverse into the neighboring cells independent of gap junctions and even can pass the blood brain barrier by diffusion, it induces a significant bystander effect (Kerr et al 1984; Fischer et al 2004). Because of its unique properties, CD/5-FC system has been used for the treatment of different types of cancer, such as colon carcinoma, glioma and pancreatic cancer (Deng et al 2011; Kaliberov et al 2007; Kaliberova et al 2008). Having been combined with radiotherapy, CD/5-FC has shown quite a promising result due to the radio-sensitizing effect of 5-FU on the treated cells (Nagy et al 2002).…”

“…The first drawback is due to the presence of normal flora in the gut which is able to convert 5-FC to 5-FU resulting in side effects. Several groups have worked to create bCD mutants in order to improve its kinetic showing promising results (Deng et al 2011; Kaliberov et al 2007; Johnson et al 2011). Yeast cytosine deaminase is another form of the enzyme which provides a few advantages over bCD including high affinity and better prodrug conversion kinetics.…”

Molecular chemotherapy of pancreatic cancer using novel mutant bacterial cytosine deaminase gene

“…CD Conversion Assay Enzyme activity of bCDwt and bCD-D314A in pancreatic cancer cells was determined by measuring the conversion of [ 3 H]5-FC to [ 3 H]5-FU by TLC as described previously (13). Clonogenic Survival Assay At 24 h after infection with 50 TCID 50 /cell of AdbCDwt or AdbCD-D314A, cells were trypsinized and diluted to the appropriate cell density into six-well culture plates and allowed to adhere overnight.…”

“…The replicationdeficient AdbCD-D314A vector encoding the mutant bCD-D314A gene driven by the cytomegalovirus promoter was developed, propagated, and titered as described previously (13).…”

Delayed Progression of Lung Metastases Following Delivery of a Prodrug-activating Enzyme

“…Several mutants have been created for an enhanced conversion rate of 5-FC to 5-FU (18)(19)(20). A triple-mutant (bCD 1525 ) displaying high specificity for 5-FC (21) exhibited increased antitumor activity and also bystander effect, a phenomenon by which the cytotoxic metabolite is transferred to nearby cells via gap junction intercellular communications (22,23).…”